Biomedical subjects

M A Quilliam

Publications and source records attributed to M A Quilliam.

At least 19 recordsLinked to original sources

PubMed2001

First report of the cyanobacterial toxin cylindrospermopsin in New Zealand.

Cylindrospermopsin, a hepatotoxin produced by cyanobacteria, has been unambiguously detected in cyanobacteria collected from a recreational lake in the Wellington region of New Zealand. To our knowledge this is the first report of cylindrospermopsin in New Zealand. Cylindrospermopsin and several microcystins were identified by reversed phase high-performance liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) using an atmospheric-pressure ionization source and an ionspray interface. The presence of cylindrospermopsin in New Zealand highlights the risk of direct cyanobacterial toxin exposure in recreational waters.

Alkaloids↗

PubMed2001

Characterization of spirolides a, c, and 13-desmethyl c, new marine toxins isolated from toxic plankton and contaminated shellfish.

Three additional marine toxins, spirolides A (1), C (3), and 13-desmethyl-C (7), were isolated from contaminated scallops and phytoplankton collections obtained from a Nova Scotian aquaculture site, as well as from batch cultures of the dinoflagellate Alexandrium ostenfeldii obtained as a single-cell isolate from these phytoplankton assemblages. The structures of these new spirolide derivatives, characterized by mass spectrometry and NMR, indicate a close relationship with spirolides B (2) and D (4) isolated previously from contaminated shellfish in the same area. All of these compounds display "fast-acting" toxicity in the traditional bioassay used for monitoring shellfish, and this is related to the presence of a cyclic imine function in all these compounds. Those spirolides containing a vicinal dimethyl group in the seven-membered ring are resistant to oxalic acid hydrolysis, whereas those that do not are readily hydrolyzed. These observations suggest that the extra methyl group on the seven-membered imine ring of 3, 4, and 7 appears to block the process of imine hydrolysis perhaps by stereochemical interference.

Animals↗

PubMed1999

Winter accumulation of paralytic shellfish toxins in digestive glands of mussels from Arcachon and Toulon (France) without detectable toxic plankton species revealed by interference in the mouse bioassay for lipophilic toxins.

Since January 1993, neurological symptoms and rapid deaths (5 to 10 min) were typically observed in the mouse bioassay of acetone extracts of digestive glands from Arcachon and Toulon (France) during the winter season. It was assumed initially that a new lipophilic toxin was present because tests using the AOAC mouse bioassay for paralytic shellfish toxins on acid extracts of whole shellfish meat were negative, no known lipophilic toxins were detected and no toxic phytoplankton species were observed in the area during the poisoning events. In this study, however, preparative isolation of the toxic factor from toxic mussel digestive glands has revealed the presence of paralytic shellfish toxins, the principal ones being gonyautoxins-2 and -3 at Arcachon and gonyautoxins-1, -4, -2 and -3 at Toulon. The toxin concentrations recorded were below levels harmful to consumers and therefore represent a false positive in the mouse bioassay for lipophilic toxins based upon acetone extraction. The origin of the toxins remains to be determined.

Animals↗

PubMed1999

Spirolide composition of micro-extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellate Alexandrium ostenfeldii.

A novel micro-extraction technique was applied to the extraction of biologically active macrocyclic imines known as spirolides from pooled individual cells isolated from spirolide-rich plankton material. For comparison, this method was also applied to pooled individual cells isolated from a unialgal culture of the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech & Tangen, a species known to produce spirolides. Both athecate cells and motile forms of gonyaulacoid dinoflagellates derived from size-fractionated plankton material from Nova Scotia, Canada were sorted and pooled by the glass micropipette isolation technique and by flow cytometry. The development of a highly sensitive analytical method for spirolides (detection limit 2 ng ml(-1) for spirolide B) using liquid chromatography-mass spectrometry (LC-MS) and application to micro-extracted samples allowed the accurate determination of spirolide composition in as few as 50 cells. Total spirolide concentrations (fmol cell(-1)) calculated from pooled micropipette isolated cells were very consistent with those based upon bulk- or micro-extractions of A. ostenfeldii cells from unialgal batch cultures in exponential growth phase. The results of the pooled cell selection from field material from two sites in Nova Scotia confirmed the association of spirolides with vegetative cells of A. ostenfeldii and related athecate forms. Combining these techniques represents a highly sensitive method for the analysis of marine toxins within complex plankton matrices, even when the toxigenic species is in low abundance, by enrichment of the target organism.

Animals↗

PubMed1999

Further studies on the analysis of DSP toxin profiles in galician mussels.

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the "DTX3" complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.

Animals↗

PubMed1998

Improved method for preparation and use of 9-anthryldiazomethane for derivatization of hydroxycarboxylic acids. Application to diarrhetic shellfish poisoning toxins.

Application of a method for the "in situ" generation of 9-anthryldiazomethane (ADAM) to the derivatization of the carboxyl function in diarrhetic shellfish poisoning (DSP) toxins revealed the formation of artifact products. Using liquid chromatography-mass spectrometry, it was determined that these artifacts were due to base-catalyzed reactions between the solvent, ethyl acetate, and the hydroxyl groups of the analyte to produce O-acetylated ADAM derivatives. Using a new formulation, with tetrahydrofuran as solvent, it was possible to eliminate these artifact reactions. Various reaction parameters have also been re-optimized to ensure quantitative derivatizations. An assessment method was developed that was useful not only for optimizing reaction parameters, but also for evaluating the reagent potency before use on important samples. Finally, application of the method to the determination of DSP toxins in plankton and mussel tissue was demonstrated.

Anthracenes↗

PubMed1998

Analysis of domoic acid in shellfish by thin-layer chromatography.

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol-acetic acid-water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 microg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay.

Animals↗

PubMed1997

Analysis of domoic acid and isomers in seafood by capillary electrophoresis.

Methods for the analysis of domoic acid (DA) based upon capillary electrophoresis (CE) combined with UV absorbance detection were investigated. DA could be analyzed using bare fused-silica capillaries in either the cationic or anionic mode with acidic or basic buffer systems, respectively. Highest performance, in terms of both separation efficiency and analysis time, was achieved with phosphate or borate buffers at a pH of approximately 9. The addition of beta-cyclodextrin to the borate buffer permitted a separation of DA and several of its isomers (isodomoic acids) that was superior to that achieved with liquid chromatography (LC). The optimum background electrolyte for the separation was 22.5 mM sodium tetraborate at pH 9.2 with mM beta-cyclodextrin. In addition, an extraction and clean-up procedure was developed and tested with mussels, clams and anchovies. Aqueous methanol extraction of samples followed by a tandem strong anion and strong cation exchange clean-up provided an extract that was completely compatible with CE analysis. A mass detection limit of 3 pg of DA injected and a method detection limit of 150 ng/g in tissues could be achieved. Comparison with LC showed that comparable precision and accuracy could be attained by the two techniques.

Animals↗

PubMed1997

Comparative toxicity of the diarrhetic shellfish poisons, okadaic acid, okadaic acid diol-ester and dinophysistoxin-4, to the diatom Thalassiosira weissflogii.

Quantitative structure-activity relationships were determined for the diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), OA diol-ester and dinophysistoxin-4 (DTX-4), using a sensitive bioassay procedure with the diatom Thalassiosira weissflogii. OA diol-ester was found to be nearly as toxic as OA. This result contradicted the accepted idea that only the free acid toxins, such as DTX-1 and OA, are potent phosphatase inhibitors. Postassay analyses using liquid chromatography-mass spectrometry (LC-MS) of cultures incubated with OA diol-ester showed that the ester had partially decomposed to OA, which explained some but not all of the observed toxicity. The formation of OA during the bioassay raised the possibility that cells exposed to inactive DSP toxin esters could metabolically activate them. This was examined in an additional experiment which showed that the hydrolysis of both DTX-4 and OA diol-ester was spontaneous and apparently not mediated by the presence of T. weissflogii cells. However, cells of T. weissflogii challenged with OA diol-ester rapidly metabolized most of the toxin to a more water-soluble product. From interpretation of mass spectral data obtained using ion-spray LC-MS, the metabolite was identified as an oxygenated diol-ester of OA, implying that it was the product of a monooxygenase-detoxification pathway. It is postulated that OA diol-ester, as a lipid-soluble, uncharged molecule with a propensity to hydrolyse to OA, may facilitate the transfer of OA across cell walls and membranes.

Animals↗

PubMed1996

Simultaneous occurrence of diarrhetic and paralytic shellfish poisoning toxins in Spanish mussels in 1993.

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.

Animals↗

PubMed1995

Molecular geometries of dibenzothiazepinone and dibenzoxazepinone calcium antagonists.

A number of dibenzothiazepinones and dibenzoxazepinones have been designed, synthesized and evaluated as calcium antagonists. Molecular geometries of these dibenzotricyclic calcium antagonists have been studied using X-ray crystallography, molecular modeling and two-dimensional NMR spectroscopy. X-Ray diffraction reveals dibenzothiazepinone 1 and dibenzoxazepinone 2 to have, respectively, flexure angles of 108 degrees and 116.9 degrees between the two benzene rings. The molecular mechanics-optimized geometry of dibenzothiazepinone 1 shows a 7 degrees smaller flexure angle than the X-ray crystallographic result, while that of dibenzoxazepinone 2 has an angle only 2 degrees smaller than the X-ray result. AM1 and ab initio calculations show that the side chains can affect the geometry of the tricyclic nucleus and both 1 and 2 have negative electrostatic potentials around the bridged portion of the tricyclics. Two-dimensional NOESY NMR spectroscopy supports the extended geometry of the 6 carbon spacer as obtained from X-ray crystallography and molecular mechanics calculations. Vasorelaxation properties among these compounds appear to be relatively insensitive to the flexure angle and to chain length. Vasorelaxation is profoundly influenced by the nature of the basic terminal moiety.

Animals↗

PubMed1994

Investigation of derivatization reagents for the analysis of diarrhetic shellfish poisoning toxins by liquid chromatography with fluorescence detection.

Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz: 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.

Animals↗

PubMed1993

Determination of erythromycin A by liquid chromatography and electrochemical detection, with application to salmon tissue.

The chromatographic performance of erythromycin A (EA) is improved significantly over that achieved on polymeric columns by using a sterically shielded octyldiisopropylsilica (Zorbax Rx-C8) column and a neutral mobile phase consisting of 5 mM aqueous sodium perchlorate-acetonitrile (50:50). This mobile phase facilitates electrochemical detection of EA at the 3-pmol level. Temperature control of both column and detection cell is important for minimizing detector noise and drift. A clean-up procedure, based on aminopropylsilica solid-phase extraction, allows the detection of EA in salmon flesh down to the 0.2-ppm level. Some of the metabolites of EA that retain the tertiary amine may also be detected by this method.

Animals↗

PubMed1993

Analysis of paralytic shellfish poisoning toxins by automated pre-column oxidation and microcolumn liquid chromatography with fluorescence detection.

Periodate oxidation of the toxins responsible for paralytic shellfish poisoning (PSP) yields fluorescent purines suitable for trace analysis by reversed-phase LC. Mobile phases containing perfluorinated acids, such as heptafluorobutyric acid, as ion-pair agents were found to provide high capacity factors for the oxidized products. Gradient elution on a microbore column with large volume injections and fluorescence detection permitted the detection of femtomole quantities of PSP toxins. A fully automated pre-column oxidation procedure was developed for an LC autosampler system in order to improve precision and allow unattended analyses. The complete method was applied successfully to various samples, including shellfish and toxic phytoplankton.

Autoanalysis↗

PubMed1993

Characterization of the oxidation products of paralytic shellfish poisoning toxins by liquid chromatography/mass spectrometry.

Periodate oxidation of the toxins responsible for paralytic shellfish poisoning (PSP) yields fluorescent products that are useful for trace analysis by reversed-phase liquid chromatography. Some toxins are oxidized to the same product, while others are transformed to a mixture of products; in all cases, the products are too unstable to be isolated preparatively. A mobile phase containing heptafluorobutyric acid as an ion-pair agent was developed to improve compatibility with liquid chromatography/mass spectrometry (LC/MS) analysis. Several LC/MS techniques were investigated: chemical ionization with the moving belt interface, atmospheric-pressure chemical ionization, thermospray, continuous-flow fast-atom bombardment, and ion-spray. Only LC/ion-spray MS was able to provide spectra of the oxidized products. The products were characterized as purines resulting from: (a) breakage of the C4-C12 bond and aromatization of the remaining rings (loss of 4 hydrogens); (b) partial elimination of a carbamoyl or N-sulfocarbamoyl side-chain (this is promoted by the presence of an N-1-hydroxy group); and (c) partial hydrolysis of an N-sulfocarbamoyl side-chain to a carbamoyl group.

Gas Chromatography-Mass Spectrometry↗

PubMed1992

Synthesis, characterization, and Ca2+ antagonistic activity of diltiazem metabolites.

Diltiazem is a calcium antagonist widely used in the treatment of angina and hypertension. The contributions of metabolites of diltiazem to the vasorelaxant effects of diltiazem were investigated. The synthesis and spectroscopic characterization of eight major cis-diltiazem metabolites are described. Three of the compounds--N, O-didemethylated metabolite (21), O-demethylated metabolite (22), and diltiazem N-oxide (27)--have been recently reported and have not previously been synthesized. The identities of all eight synthetic metabolites have been verified with samples obtained from human urine using combined LC-MS/MS. The Ca2+ antagonistic activities of diltiazem and its metabolites (except 27) were studied on hamster aorta preparations depolarized with KCl. The order of potencies (IC50 +/- SE, microM) is as follows: diltiazem (0.98 +/- 0.47) greater than 17 (2.46 +/- 0.38) greater than or equal to 23 (3.27 +/- 1.02) greater than 26 (20.2 +/- 10.5) greater than 22 (40.4 +/- 15.4) greater than or equal to 25 (45.5 +/- 18.1) greater than 21 (112.2 +/- 33.2) greater than or equal to 24 (126.7 +/- 24.2). Structure-activity relationships are also discussed.

Animals↗

PubMed1992

Determination of erythromycin A in salmon tissue by liquid chromatography with ion-spray mass spectrometry.

A reverse-phase liquid chromatography/mass spectrometry (LC/MS) method, incorporating gradient elution, is described for the characterization of residual erythromycin A and its metabolites in salmon tissue. The method uses ion-spray, a mild atmospheric pressure ionization technique which provides an abundant protonated molecule well suited for selected ion monitoring experiments. Tandem mass spectrometry (MS/MS) using collision-induced dissociation was used to provide structural information. The LC/MS method was tested for the analysis of salmon tissue spiked with erythromycin A at levels between 0.01 and 1 p.p.m. A simple extraction and clean-up procedure, slightly modified from that described by Takatsuki et al. (J. Assoc. Off. Anal. Chem. 70, 708 (1987)), was used in this work. Using selected ion and selected reaction monitoring techniques, the LC/MS and LC/MS/MS methods provided detection limits of < 10 and 50 ng g-1, respectively. Confirmatory full-scan LC/MS and LC/MS/MS spectra were obtained at the 0.5 and 1 microgram g-1 levels, respectively. Using a combination of these techniques, the presence of residual erythromycin A was confirmed in the tissue of fish administered medicated feed containing the antibiotic. In addition, several metabolites and degradation products of erythromycin A, including anhydro-erythromycin and N-demethyl-erythromycin, were detected and where possible confirmed by comparison with authentic compounds. Although this analytical method has been shown to afford the necessary sensitivity and precision, application of these techniques to high-throughput quantitative analyses will require development of an improved clean-up procedure and preferably also of a suitable surrogate internal standard.

Animals↗

PubMed1992

Ionspray mass spectrometry of marine toxins. IV. Determination of diarrhetic shellfish poisoning toxins in mussel tissue by liquid chromatography/mass spectrometry.

An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10 ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.

Animals↗